CN104023732A - Prophylactic or therapeutic agent for oral diseases - Google Patents

Abstract

The present invention provides a preventive and/or therapeutic agent for oral diseases, which is obtained from Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri and Streptococcus mitis ) and have one or more lactic acid bacteria selected from the following (1) to (6) as active ingredients, (1) do not have the ability to produce volatile sulfur compounds (VSC); (2) Does not have the ability to produce water-insoluble glucan; (3) has adhesion to the tooth surface and/or oral cavity cells; (4) has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria; (5) No pathogenicity of infective endocarditis; (6) no dental caries. The present invention can inhibit the production of volatile sulfur compounds caused by oral bacteria, has no caries and infective endocarditis pathogenicity, and can effectively and safely prevent and/or treat oral diseases.

Description

Prophylactic or therapeutic agent for oral diseases

technical field

The invention relates to a preventive or therapeutic agent for oral diseases such as dental caries, periodontal disease, halitosis and other oral diseases or discomfort symptoms, which can prevent, improve and/or treat.

Background technique

From an invisible aesthetic point of view, bad breath plays an important role in affecting a comfortable personal and social life. According to the report of quantitative testing on the impact of bad breath on oral QOL (quality of life: quality of life), this impact is even greater than the morphological aesthetic factors (non-patent literature 1). Moreover, in Japan, which is rapidly entering an aging society, bad breath may make it more difficult to care for the elderly (Non-Patent Document 2).

The main cause of bad breath is three kinds of volatile sulfur compounds (VSC), hydrogen sulfide, methyl mercaptan, and dimethyl sulfide (Non-Patent Documents 3 and 4). VSC is produced when cysteine, methionine, etc. contained in epithelial cells and white blood cell residues peeled off in the oral cavity, food, etc. are decomposed by oral bacteria. According to reports (Non-Patent Document 5), periodontal pathogenic bacteria such as Fusobacterium, Porphyromonas, Veillonella parvum and Helicobacter can produce a large amount of VSC in a culture test using oral bacteria. In addition, Lactobacillus olis isolated from the oral cavity also has a strong ability to produce VSC. According to data report (non-patent document 6) in addition, VSC is except that itself has strong bad smell, also has toxicity to living tissue in low concentration state, so it is not only a kind of inducement material that forms halitosis, also very May be a factor that contributes to the exacerbation of periodontal disease. Therefore, inhibiting the VSC produced by oral bacteria, especially periodontal pathogenic bacteria, and preventing or improving halitosis can not only improve QOL, but also have very important significance in maintaining oral health.

In recent years, the probiotic technology has also been tried to be used in the oral cavity. For example, it has been reported that Lactobacillus salivarius (Lactobacillus salivarius) TI2711 strain (Patent Document 1) is effective in halitosis, dental caries, periodontal disease, and oral infection. Etc. effective.

prior art literature

patent documents

Patent Document 1: Japanese Invention Patent No. 4203855

non-patent literature

Non-Patent Document 1: Japanese Public Health Journal 49, 2002, p298

Non-Patent Document 2: Survey and Research Report of (Financial) Medical Economics Research Institute Heisei 7

Non-Patent Document 3: Arch Oral Biol16, 1971: 587-597

Non-Patent Document 4: Int. Dent. J28, 1978: 309-319

Non-Patent Document 5: Journal of Oral Hygiene 51, 2001, p778-792

Non-Patent Document 6: Journal of Niigata Dental Society 32, 2002, p309-310

Non-patent literature 7: J Med Microbiol39, 1991: 179-182

Non-Patent Document 8: Eur J Clin Microbiol Infect Dis24, 2005: 31-40

Non-Patent Document 9: Journal of Infection53, 2006: e5-e10

Contents of the invention

The problem to be solved by the invention

On the other hand, bacteria of the genus Lactobacillus and Streptococcus in the oral cavity are considered to be one of the causes of dental caries and infective endocarditis. Streptococcus sobrinus is known as a dental caries bacterium, and its harmfulness surpasses that of Streptococcus mutans, and it is a bacterium that has attracted attention as a cause of dental caries in the clinical field. In addition, according to reports, the most frequently detected microorganisms causing infective endocarditis are Streptococcus bacteria in the oral cavity such as Streptococcus sanguinis and Streptococcus oralis (Non-Patent Document 7) , Lactobacillus bacteria also have endocarditis-inducing strains (Non-Patent Documents 8 and 9).

Therefore, it is desirable that the bacteria used to improve the oral flora can inhibit the production of VSC and have no caries and infective endocarditis pathogenicity, but no safe bacteria with these properties have been reported so far. strains.

Therefore, the object of the present invention is to provide a novel lactic acid bacterial strain and a preventive or therapeutic agent for oral diseases using the strain to prevent, improve and/or treat oral diseases or discomfort symptoms, which can inhibit volatile sulfur caused by oral bacteria. The compound is produced, and has no carious and infective endocarditis pathogenicity, and is safe in the oral cavity.

Solution to the problem

The inventors have found through concentrated research on the microorganisms in the oral cavity that they belong to specific bacteria such as Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri, and Streptococcus mitis. Lactic acid bacteria have very high adhesion to the tooth surface and oral cavity cells, which can hinder the proliferation of periodontal pathogenic bacteria that cause bad breath, and can inhibit the production of VSC, and the lactic acid bacteria themselves will not produce VSC and water-insoluble glucan. In addition, they have further found that the lactic acid bacteria are useful bacteria for improving oral flora without caries and infective endocarditis pathogenicity, thereby completing the present invention.

That is to say, the present invention provides a preventive or therapeutic agent for oral diseases, which is obtained from Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri and Streptococcus mitis ( One or more lactic acid bacteria selected from Streptococcus mitis) and having all the following properties (1) to (6) are used as active ingredients.

(1) Does not have the ability to produce volatile sulfur compounds (VSC)

(2) Does not have the ability to produce water-insoluble dextran

(3) Adhesive to tooth surface and/or oral cavity cells

(4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria

(5) No pathogenicity of infective endocarditis

(6) No caries

In addition, the present invention also provides an agent for preventing and/or improving bad breath, which is obtained from Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri and Streptococcus mitis mitis) and have one or two or more lactic acid bacteria selected from the following (1) to (6) as active ingredients.

(1) Does not have the ability to produce volatile sulfur compounds (VSC)

(2) Does not have the ability to produce water-insoluble dextran

(3) Adhesive to tooth surface and/or oral cavity cells

(4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria

(5) No pathogenicity of infective endocarditis

(6) No caries

In addition, the present invention provides a lactic acid bacterium named as Lactobacillus crispatus (Lactobacillus crispatus) YIT12319, submitted for deposit as FERM BP-11500, named as Lactobacillus fermentum (Lactobacillus fermentum) YIT12320, submitted as FERM BP-11501 Deposited lactic acid bacteria named as Lactobacillus gasseri (Lactobacillus gasseri) YIT12321, submitted for deposit as FERM BP-11502, or named Streptococcus mitis (Streptococcus mitis) YIT12322, submitted for deposit as FERM BP-11503 Lactic acid bacteria.

Moreover, this invention provides the food-drinks containing the said lactic acid bacteria.

At the same time, the present invention also provides an oral composition containing the above-mentioned lactic acid bacteria.

In addition, the present invention provides that in the manufacture of prophylactic or therapeutic agents for oral diseases, use Lactobacillus crispatus (Lactobacillus crispatus), fermented Lactobacillus (Lactobacillus fermentum), Lactobacillus gasseri (Lactobacillus gasseri) and the chain One or more lactic acid bacteria selected from Streptococcus mitis and possessing all of the following properties (1) to (6).

(1) Does not have the ability to produce volatile sulfur compounds (VSC)

(2) Does not have the ability to produce water-insoluble dextran

(3) Adhesive to tooth surface and/or oral cavity cells

(4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria

(5) No pathogenicity of infective endocarditis

(6) No caries

In addition, the present invention provides that, in the manufacture of halitosis prevention and/or improving agent, use Lactobacillus crispatus (Lactobacillus crispatus), fermented Lactobacillus (Lactobacillus fermentum), Lactobacillus gasseri (Lactobacillus gasseri) and alleviating disease chain One or more lactic acid bacteria selected from Streptococcus mitis and possessing all of the following properties (1) to (6).

(1) Does not have the ability to produce volatile sulfur compounds (VSC)

(2) Does not have the ability to produce water-insoluble dextran

(3) Adhesive to tooth surface and/or oral cavity cells

(4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria

(5) No pathogenicity of infective endocarditis

(6) No caries

In addition, the present invention also provides a method for preventing and/or treating oral diseases, which is obtained from Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri and Streptococcus mitis ( Streptococcus mitis) with an effective amount of 1 or 2 or more lactic acid bacteria selected from the following (1) to (6) and given to the patient.

(1) Does not have the ability to produce volatile sulfur compounds (VSC)

(2) Does not have the ability to produce water-insoluble dextran

(3) Adhesive to tooth surface and/or oral cavity cells

(4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria

(5) No pathogenicity of infective endocarditis

(6) No caries

In addition, the present invention also provides a method for preventing and/or improving bad breath, which is obtained from Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus gasseri (Lactobacillus gasseri) and Streptococcus mitis Streptococcus mitis) with an effective amount of 1 or 2 or more lactic acid bacteria selected from the following (1) to (6) and given to the patient.

(1) Does not have the ability to produce volatile sulfur compounds (VSC)

(2) Does not have the ability to produce water-insoluble dextran

(3) Adhesive to tooth surface and/or oral cavity cells

(4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria

(5) No pathogenicity of infective endocarditis

(6) No caries

In addition, the present invention also provides a lactic acid bacterium for preventing and/or treating oral diseases. One or two or more species selected from Streptococcus mitis and having all of the following properties (1) to (6).

(1) Does not have the ability to produce volatile sulfur compounds (VSC)

(2) Does not have the ability to produce water-insoluble dextran

(3) Adhesive to tooth surface and/or oral cavity cells

(4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria

(5) No pathogenicity of infective endocarditis

(6) No caries

The present invention also provides a lactic acid bacterium for preventing and/or improving bad breath, the lactic acid bacterium is obtained from Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri and Streptococcus mitis (Streptococcus mitis) selected from one or more of the following properties (1) to (6).

(1) Does not have the ability to produce volatile sulfur compounds (VSC)

(2) Does not have the ability to produce water-insoluble dextran

(3) Adhesive to tooth surface and/or oral cavity cells

(4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria

(5) No pathogenicity of infective endocarditis

(6) No caries

In addition, the present invention also provides a primer or probe, which is directed against the nucleotide sequence selected from SEQ ID NO: 4-8 or Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 formed by complementarily arranging the nucleotide sequence. (FERM BP-11500) is specific.

In addition, the present invention also provides a pair of primers for the base sequence selected from SEQ ID NO: 4 and 8, SEQ ID NO: 5 and 8, SEQ ID NO: 6 and 8, and SEQ ID NO: 7 and 8 or by Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500) formed by complementary arrangement on the base sequence has specificity.

And the present invention also provides a detection method of Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500) characterized by using the above primers, primer pairs or probes.

The effect of the invention

The lactic acid bacteria of the present invention can stay firmly on the tooth surface and in the oral cavity, and have the effect of hindering the proliferation of periodontal pathogenic bacteria such as dental caries pathogenic bacteria and halitosis-causing bacteria. On the other hand, they have no ability to produce VSC or water-insoluble glucan , without the pathogenicity of dental caries and infective endocarditis, so it can be effectively used to improve the health of the oral flora, prevent dental caries, periodontal disease, bad breath and other oral diseases or discomfort symptoms, Drugs, food and beverages, pet foods, and oral compositions for improving or treating effects.

Description of drawings

FIG. 1 is a graph showing changes in hardness of bovine tooth enamel exposed to YIT12319-12321.

Fig. 2 is a graph showing the amount of water-insoluble glucan in biofilm recovered from bovine tooth enamel contacted with YIT12319-12321.

Fig. 3 is a graph showing changes in hardness of bovine tooth enamel exposed to YIT12322.

Fig. 4 is a graph showing the amount of water-insoluble glucan in biofilm recovered from bovine tooth enamel contacted with YIT12322.

Fig. 5 is a graph showing changes in hardness of bovine tooth enamel exposed to YIT12321.

Fig. 6 is a graph showing the sucrose assimilability of YIT12321.

Fig. 7 is a graph showing DNA bands amplified by specific primer pairs on YIT12319 (the arrows indicate specific DNA bands, and the primer pairs used next to them).

Detailed ways

As the lactic acid bacteria used in the present invention, there are lactic acid bacteria strains belonging to Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri and Streptococcus mitis, and may be One or more of the above strains.

Specifically, it was named as Lactobacillus crispatus (Lactobacillus crispatus) YIT12319, and was deposited as FERM BP-11500 on April 28, 2011 (the same below) in the Patent Biological Depositary Center of the Product Evaluation Technical Foundation of the Independent Administrative Legal Person (Address: Ibaraki The lactic acid bacteria of 1-1-1 Higashi, Tsukuba City, Central No. 6) is named as Lactobacillus fermentum YIT12320, and the lactic acid bacteria preserved as FERM BP-11501 is named as Lactobacillus gasseri (Lactobacillus gasseri) YIT12321 , lactic acid bacteria deposited as FERM BP-11502 named Streptococcus mitis YIT12322, lactic acid bacteria deposited as FERM BP-11503, etc. The lactic acid bacteria also include offspring strains (natural mutant strains, mutant strains subjected to mutation treatment, mutant strains obtained by genetic manipulation, etc.) using the above-mentioned lactic acid bacteria as the mother strain.

Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500), Lactobacillus fermentum (Lactobacillus fermentum) YIT12320 (FERM BP-11501), Lactobacillus gasseri (Lactobacillus gasseri) YIT12321 (FERM BP-11502) and the chain Streptococcus mitis YIT12322 (FERM BP-11503), as shown in the following examples, was isolated from the human oral cavity by the inventor for the first time, and as a result of identity retrieval through the 16S-rDNA gene sequence, it was determined that they belonged to curly milk Bacillus, Lactobacillus fermentum, Lactobacillus gasseri or Streptococcus mitis. Since these lactic acid bacterial strains have the characteristic properties of the following (1) to (6), they were found to be novel strains.

(1) Does not have the ability to produce volatile sulfur compounds (VSC)

(2) Does not have the ability to produce water-insoluble dextran

(3) Adhesive to tooth surface and/or oral cavity cells

(4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria

(5) No pathogenicity of infective endocarditis

(6) No caries

In addition, Lactobacillus gasseri (Lactobacillus gasseri) YIT12321 (FERM BP-11502) was also confirmed to have the following properties (7).

(7) Not assimilated with sucrose

In this specification, (1) "does not have the ability to generate volatile sulfur compounds (VSC)" means that in the test (test example 2) shown in the following examples, the ability to generate VSC is +/-(H ₂ S < 0.70 μg/10 ml/OD, CH ₃ SH < 1.17 μg/10 ml/OD), better case - value (below the lower limit of detection). Lactic acid bacteria having this property (1) are satisfactory in that they do not produce substances that cause bad breath and do not produce VSC, which is a factor that can aggravate periodontal disease.

In addition, in this specification, (2) "not having the ability to produce water-insoluble glucan" means that in the test (Test Example 3) shown in the following examples, the ability to produce water-insoluble glucan It is +/- (bacteria produce attachment), and the better case is - (no formation). Lactic acid bacteria having the property (2) are satisfactory in that they do not produce water-insoluble glucans that cause dental caries.

In addition, in this specification, (3) "has adhesion to the tooth surface" means that in the test (Test Example 5) shown in the following examples, the adhesion rate to S-HA is 1.0% or more, A better case is to reach more than 10%. In addition, in this specification, (3) "has adhesiveness to oral cavity cells" means that in the test (test example 6) shown in the following example, the adhesiveness to oral cavity cells is Total30cells/0.16 ^mm2 or more. Among the properties (3), lactic acid bacteria having adhesiveness to tooth surfaces or cells in the oral cavity are satisfactory because they are easily fixed in the oral cavity even if the residence time is short when they are ingested orally. From the same point of view, lactic acid bacteria that have adhesion to tooth surfaces or cells in the oral cavity are more ideal.

In addition, in this specification, (4) "has the effect of inhibiting the proliferation of halitosis-causing bacteria and/or periodontal pathogenic bacteria" means that in the test (test example 7) shown in the following examples, the diameter of the antibacterial ring (diameter ) totals more than 9mm. Lactic acid bacteria having this property (4) are desirable in that they have excellent preventive and therapeutic effects on halitosis and/or periodontal disease.

In addition, in this specification, (5) "no pathogenicity of infective endocarditis" means that in the test (test example 8) shown in the following examples, the pathogenicity of mouse endocarditis was negative . Lactic acid bacteria having this property (5) are extremely safe and are very suitable for use in food and beverages, oral compositions, and the like.

In addition, in this specification, (6) "no caries" means that in the test (Test Example 9) shown in the following examples, the hardness reduction rate of bovine tooth enamel was not equal to that of the test using an artificial oral cavity device. to 5%. Lactic acid bacteria having this property (6) are satisfactory in that they do not produce water-insoluble glucan that causes dental caries and do not detach enamel.

In addition, in this specification, (7) "not assimilated with sucrose" means that sucrose cannot be used as a growth substrate of bacteria in the test (Test Example 9) shown in the following examples. Lactic acid bacteria having this property (7) are desirable in that they are less likely to produce lactic acid and can reduce the risk of dental caries.

In the present invention, the thallus of the above-mentioned lactic acid bacteria can not only use the product isolated from the culture obtained according to the common lactic acid bacteria culture method through centrifugation and other bacterial collection methods, but also can use the culture of lactic acid bacteria itself after the end of the culture. Or use a concentrate obtained by concentrating the culture medium. In addition to live bacteria, processed bacterial cells can also be used. The treated product is not particularly limited as long as it is processed by a common method, for example, heat treatment, drug treatment with antibiotics, etc., treatment with chemical substances such as formalin, ultraviolet treatment, gamma rays, etc. Radiation-treated dead bacteria, their freeze-dried products, cultures containing the above, etc.; bacterial lysates generated by ultrasonic waves, enzyme-treated bacterial liquids, and separation from these liquids by solid-liquid separation methods such as filtration or centrifugation The solid residues, etc. that come out; the treatment solution that removes the cell wall by enzyme or mechanical method, the concentrate of the treatment solution, their dilution, their dry matter, etc.; after the bacteria are dissolved by surfactants, etc., they are precipitated by ethanol, etc. The nucleic acid containing components obtained afterward; the products obtained by separating and purifying the above-mentioned bacterial lysates or enzyme-treated cell fluids generated by ultrasonic waves or the like by various methods such as chromatography.

The medium for culturing lactic acid bacteria is not particularly limited, and various mediums can be used.

For example, carbon sources such as glucose, fructose, galactose, sucrose, inorganic salts such as monopotassium phosphate, dipotassium hydrogen phosphate, magnesium sulfate, sodium sulfite, sodium thiosulfate, ammonium phosphate, polyvalent peptone, yeast extract, corn Organic nutrient sources such as pulp, and in addition to the nutrient medium for the growth of lactic acid bacteria that are usually added with various amino acids, vitamins, etc. as needed, milk culture medium containing milk can also be used.

As for the cultivation, it is only necessary to satisfy the condition that the bacteria grow well. There are no special restrictions on the culture method, such as aeration culture, anaerobic culture, stirring culture, shaking culture, static culture, etc., but considering productivity, static culture under aerobic conditions is the most ideal.

In addition, the culture temperature is usually 10-50°C, preferably 25-37°C; the culture time is usually 6 hours to 3 days, preferably 8 hours to 3 days.

In addition, the pH (25°C) of the culture medium is 3-10, preferably 5-8. For example, organic acid salts such as carbonic acid, acetic acid, citric acid, fumaric acid, malic acid, lactic acid, gluconic acid, and tartaric acid, inorganic salts such as phosphoric acid, hydrochloric acid, and sulfuric acid, and sodium hydroxide can be used as buffers for adjusting the pH of the medium. Hydroxide, ammonia, ammonia water, etc. may be used alone or in combination of two or more.

The "oral disease" targeted by the oral disease prevention and/or treatment agent of the present invention refers to oral diseases caused by pathogenic bacteria in the oral cavity such as dental caries pathogenic bacteria, periodontal pathogenic bacteria, and candida, such as dental caries, gingivitis, dental Periodontal disease such as periodontal disease, thrush, etc. Among them, oral diseases with the best effect are periodontal diseases such as gingivitis and periodontitis.

Dental caries pathogens include Streptococcus mutans, cousin Streptococcus sobrinus, periodontal pathogens include Porphyromonas gingivalis, Prevotella intermedia, tartar Treponema denticola, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, etc. Bacteria that cause oral thrush include Candida albicans and the like.

The "halitosis-causing bacteria" targeted by the halitosis prevention and/or improvement agent of the present invention include Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Treponema denticola, Veillonella dispar, etc.

As shown in the following examples, the lactic acid bacteria of the present invention have adhesiveness to the tooth surface and the inner surface of the oral cavity. intermedia) and Aggregatibacter actinomycetemcomitans have the effect of hindering proliferation. In addition, the lactic acid bacteria of the present invention do not produce VSCs that cause bad breath and water-insoluble glucans that cause dental caries, do not cause enamel loss, and do not induce infective endocarditis.

Therefore, the lactic acid bacteria of the present invention can promote the improvement of oral flora, and can be used as a treatment for various oral diseases or discomforts such as dental caries, gingivitis, periodontitis, thrush, and bad breath caused by pathogenic bacteria in the oral cavity. Drugs, food and beverages, pet foods, oral compositions, etc. that prevent, improve, or treat symptoms.

The dosage forms of oral preparations when used as medicine include lozenges, capsules, granules, dragees, pills, granules, powders, powders, sustained-release preparations, suspensions, emulsions, syrups, freeze-dried preparations, liquids, Elixirs etc.

The above-mentioned formulations can be produced by common methods, and the lactic acid bacteria of the present invention can be used alone or in combination with approved drug carriers. Such carriers include lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid and other excipients; starch, maltodextrin, gum arabic powder, edible gelatin, methyl Binders such as cellulose, hydroxypropyl cellulose, crystalline cellulose, ethyl cellulose, polyvinylpyrrolidone, polyethylene glycol; hydroxypropyl starch, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, carboxymethyl cellulose Disintegrants such as methyl cellulose and low-substituted hydroxypropyl cellulose; surfactants such as sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80, etc.; talcum powder, waxes, hydrogenated vegetable oil, Magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol and other lubricants; light anhydrous silicic acid, aluminum hydroxide xerogel, synthetic aluminum silicate, magnesium silicate and other fluidity promoters Diluents such as distilled water for injection, normal saline, glucose solution, olive oil, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol, etc. In addition, if necessary, commonly used flavoring agents, coloring agents, fragrances, bactericides, osmotic pressure regulators, pH regulators, emulsifiers, absorption aids, antioxidants, thickeners, tonicity agents, etc. additive.

In addition, when used as food and drink, pet food, etc., the lactic acid bacteria of the present invention may be contained in the above food and drink by adding various nutrients as necessary. Such foods and beverages can be used as health foods or food materials that have the effect of preventing or improving dental caries, periodontal disease, and bad breath, and can be marked on these foods and beverages or their containers that they have the above effects.

As for the form of food and drink, additives that can be used as food and drink can be used appropriately, and made into a form suitable for consumption by common methods, such as granules, granules, lozenges, capsules, pastes, etc., and can also be added to various A variety of foods, including processed meat products such as ham and sausage; fish cakes, cylindrical fish cakes and other aquatic products; bread, cakes, butter, milk powder, fermented food and beverages, and can also be added to water, juice, milk, soft drinks , tea drinks and other beverages used. Among these products, fermented milk containing active ingredients of lactic acid bacteria, fermented drinks (lactic acid bacteria drinks), fermented soybean milk, fermented fruit juices, fermented vegetable juices, and other fermented products, as well as nutritional supplements such as tablets and capsules, are ideal.

Fermented products can be produced using common methods. For example, fermented milk, inoculate and cultivate lactic acid bacteria in the sterilized milk medium, and then homogenize it to obtain the fermented milk base material. Next, add the separately prepared syrup solution, mix it, homogenize it with a homogenizer, etc., and add flavor to the final product. The fermented milk obtained by this method can be a product in any form such as pure type, soft type, fruit flavor type, solid state, and liquid state.

Specific forms when used as an oral composition include mouthwash, mouthwash, toothpaste, tooth powder, liquid toothpaste, oral ointment, gel, lozenge, granule, microgranule, jelly, lozenge, tablet The best forms are toothpaste, mouthwash, jelly, lozenges, lozenges, tablets, etc.

The content of the lactic acid bacteria of the present invention in the above-mentioned medicines, food and drink, pet food, oral composition, etc. is not particularly limited, and can be appropriately adjusted in combination with the daily dosage or intake. For example, when the dosage form is liquid, the concentration of lactic acid bacteria reaches 1× 10 ⁶ CFU/mL～1×10 ⁸ CFU/mL is better, and 1×10 ⁷ CFU/g～1×10 ¹⁰ CFU/g is ideal for solids.

When using the lactic acid bacteria of this invention, there are no severe restrictions on the dose or ingestion amount. If the target of use or applicable disease is different, the effect obtained will be different, so it should be set appropriately. However, the number of lactic acid bacteria should be more than 1×10 ³ CFU in the daily dose, and 1×10 3 CFU in the daily dose. The amount of 10 ³ to 1×10 ¹³ CFU is more preferable, and the amount of 1×10 ⁶ to 1×10 ¹⁰ CFU in the daily dose is the most optimal.

Through the analysis of Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 and other Lactobacillus crispatus (Lactobacillus crispatus) genomic DNA, it was found that the base sequence selected from sequence numbers 4-8 has specificity relative to Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 and if probes or primers having these base sequences were used, it was found that the gene of Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 could be specifically detected or amplified.

The probe or primer of the present invention is formed by a base sequence selected from sequence numbers 4 to 8 or arranged in a complementary manner on the base sequence, and is aimed at Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500) bacterial strain Specific probes or primers.

As the above-mentioned primer, a nucleotide sequence selected from SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 4 and 6, SEQ ID NO: 4 and 7, and SEQ ID NO: 4 and 8, or a base sequence formed by arranging complementary base sequences Primers, the higher the specificity for Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500), the better.

Using the above-mentioned probes or primers does not require complex operations such as confirmation of physiological and biochemical properties, as long as the DNA from Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500) extracted from the specimen is used for PCR reaction, the The biological type determination, analysis, detection and other operations of the strain can be performed quickly and easily.

Example

The present invention will be described more specifically through examples below, but the present invention is not limited to these examples.

Test Example 1 Isolation of Oral Bacteria

1) Collection of subjects and samples

Tartar (including saliva) and tongue coating were collected from the mouths of 56 test subjects (34 males, 22 females, 25-66 years old, average age 40.6 years old). After these oral samples were collected, they were kept on ice before use. For bacteria isolation, these samples were mixed in equal amounts, and 300 μL of them were added to 2.7 mL of sterilized anaerobic transfer medium to prepare a 10 ^-1 dilution (3.0 mL for aerobic and anaerobic culture × each 1 stick).

2) Culture and isolation of oral bacteria

In the anaerobic glove box, smear the oral cavity sample on the Brucella rabbit hemolysis plate medium (BRU plate medium, tryptophan 10.0g, paptamine 10.0g, yeast extract 2.0g, glucose 1.0g, chloride Sodium chloride 5.0g, acidic sodium sulfate 0.1g, hemoglobin 5.0mg, vitamin K 11.0mg, growth aid solution 10.0mL, hemolysis 60.0mL, agar 15.0g, pH7.0). 1.8 mL of the sterilized anaerobic dilution was dispensed into a disposable test tube (FALCON2058), and then 200 μL of the 10 ^-1 dilution was added to prepare a 10 ^-2 dilution. Follow the same method until ^10-10 dilutions are made, and then smear 100 μL of various dilutions on the plate medium, and culture according to the conditions shown in Table 1.

On the other hand, use a spiral system (automatic plate smearing device) to add 5.0% sheep blood TSA plate medium (TSA plate medium, casein peptone 15.0g, soybean peptone 5.0g, sodium chloride 5.0g, defibrated sheep blood 50.0mL, agar 15.0g, DW1000mL, pH7.3), M R S plate medium (Proteose Peptone No.3 10.0g, Beef Extract10.0g, Yeast Extract5.0g, Dextrose20.0g, Polysorbate801.0g, Ammonium Citrate2. 0g, Sodium Acetate5.0g, Magnesium Sulfate0.1g, ManganeseSulfate0.05g, Dipotassium Phosphate2.0g, Agar15g, DW1000mL), LBS plate medium (Pancreatic Digest of Casein10.0g, Yeast Extract5.0g, Monopotassium Phosphate Cit6.0g, Ammonium2 .0g, Dextrose20.0g, Polysorbate801.0g, Sodium Acetate Hydrate25.0g, Magnesium Sulfate0.575g, ManganeseSulfate0.12g, Ferrous Sulfate0.034g, Agar15.0g, Lab-lemco powder (Oxoid)8g, Sodium acetate, D00gmL0g, trihydrate105g Acetate3.7mL), and Mitis-Salivarius plate medium (MS plate medium, Bacto Tryptose10g, Bacto Proteose Peptone No.35g, Bacto Proteose Peptone5g, Bacto Dextrose1g, Bacto Saccharose50g, Dipotassium Phosphate4g, Trypan Blue0.075g, Bactolet Crystal0 0008g, Bacto Agar15g DW1000mL, pH7.0 ± 0.2) etc. after smearing samples, cultivate according to the conditions shown in Table 1.

【Table 1】

Plate culture medium and culture method

^a GB: cultured in an anaerobic glove box

^b GP: Culture was performed using AnaeroPack anaerobic system (Mitsubishi Gas Chemical)

The number of bacteria in the oral cavity propagated in various plate culture media is shown in Table 2.

In terms of MRS, MS and LBS plate medium, according to the order of the plate medium coated with high dilution rate dilution, fish bacteria from colonies with different shapes, use MRS or BHI liquid medium (calf brain extract) 12.5g, bovine heart muscle extract 5.0g, peptone 10.0g, glucose 2.0g, sodium chloride 5.0g, disodium hydrogen phosphate 2.5g, DW1000mL, pH7.4±0.2) after proliferation, in 2×BHI: Suspended in a solution of glycerol and glycerol = 1:1, stored at -80°C, and used for the following experiments.

【Table 2】

The number of bacteria in the oral cavity propagated in various plate media

*BRU: total anaerobic bacteria, TSA: total general anaerobic bacteria, MRS: lactic acid bacteria, Bifidobacterium, MS: Streptococcus genus, LBS: Lactobacillus genus

Evaluation of Test Example 2 on the ability to generate VSC

The cryopreserved strain isolated in Test Example 1 was used. In addition, Fusobacterium nucleatum (Fusobacterium nucleatum) YIT6069 was used as a positive control strain.

The above-mentioned isolated strains were inoculated into MRS liquid medium, and anaerobic culture was carried out at 37° C. for 24 hours. Take 0.04mL of the bacterium culture solution and add 1% D-(+)-glucose, 66mM DL-methionine (final concentration 500-1,500μM) to 4mL modified GAM liquid medium (5.0g peptone, 3.0g soy peptone, Show peptone 5.0g, digestive serum powder 10.0g, yeast extract powder 2.5g, meat extract powder 2.2g, liver extract powder 1.2g, glucose 0.5g, soluble starch 5.0g, L-tryptophan 0.2g, L - cysteine hydrochloride 0.3g, sodium thioglycolate 0.3g, L-arginine 1.0g, vitamin K15mg, hemoglobin 10mg, potassium dihydrogen phosphate 2.5g, sodium chloride 3.0g, DW1000mL, pH7.3) The medium was cultured again, and anaerobic culture was carried out at 37°C. 24 hours after the start of the culture, add 0.16 mL of 6.0N hydrochloric acid solution to the culture solution to lower the pH to below 1 to stop the substance metabolism of the bacteria.

Thereafter, volatile sulfur compounds (VSCs) volatilized in the headspace phase were analyzed using a gas chromatograph or an OralChroma ^TM portable gas chromatograph. A negative control was then carried out with the medium incubated anaerobically at 37°C for 24 hours.

Evaluation of Test Example 3 Ability to Produce Water-Insoluble Glucan

In BHI or MRS liquid medium, the cryopreserved strain isolated by Test Example 1 was inoculated with 2 platinum fungus, and then anaerobically cultured at 37° C. for 24 hours. Take 0.04mL of the bacterium culture solution in HI containing 1% sucrose (5.0g of bovine heart muscle extract, 10.0g of peptone, 2.0g of glucose, 5.0g of sodium chloride, 2.5g of disodium hydrogen phosphate, DW1000mL, pH7 .4±0.2), or 4mL of MRS+HI mixed (7:3) liquid medium for re-cultivation, and the test tube was inclined at 45 degrees for anaerobic culture at 37°C.

The ability to produce water-insoluble dextran was evaluated by the amount of dextran attached to the test tube wall and its attachment strength. After culturing for 24 hours, remove the culture medium from the test tube, add 4mL of PBS, and turn it gently 3 times to wash the tube wall. After removing the PBS, the amount of water-insoluble dextran attached to the test tube wall was visually judged according to the criteria in Table 3.

Table 3 shows the results of Test Examples 2 and 3. From the cryopreserved strains isolated in Test Example 1, a total of 241 strains surrounded by dotted lines in Table 3 were selected. Among them, there are 176 strains of Streptococcus bacteria isolated from MRS and MS medium, and 65 strains of Lactobacillus-like bacteria isolated from LBS medium.

In addition, as a positive control strain, Fusobacterium nucleatum (Fusobacterium nucleatum) YIT6069 produced VSC according to the amount of methionine added.

【table 3】

The ability of isolated strains to produce VSC and the ability to produce water-insoluble glucan (the numbers in the table represent the number of strains)

1. Ability to produce VSC; -, lower than the lower limit of detection +/-, (H ₂ S<0.70μg/10ml/OD, CH ₃ SH<1.17μg/10ml/OD), +, produce

2. The ability to produce non-water-soluble dextran; -, not produced, +/-, attached to the tooth body, +, produced

The biological classification of test example 4 bacterial classification is determined

DNA was extracted by the benzyl chloride method. The cultured cells were centrifuged at 830×g for 10 minutes to obtain the cells. 200 μL of DNA Extraction buffer, 400 μL of benzyl chloride, and 300 mg of glass beads (0.1 mm in diameter) were added to the cells. After shaking using the Fast Prep sample processing system (speed 6.5, 30 seconds), an equivalent amount of isopropanol was added to the supernatant obtained by centrifugation (20,000×g, 5 minutes, 4° C.) and then thoroughly stirred. After removing the supernatant by centrifugation (20,000×g, 10 minutes, 4°C), add 150 μL of 70% ethanol, air-dry the precipitate obtained after centrifugation again, and dissolve it in an appropriate amount of TE (Tris-EDTA) . As for the PCR reaction solution, in the total volume of 25 μL, make 3.2 μL of 10×PCR buffer (1×PCR buffer=10mM Tris-HCl (pH8.3), 50mM KCl, 1.5mM MgCl ₂ ), 2.5uL of 2.5 mM dNTP (deoxynucleotide triphosphate), 0.5 μL of 25 pmol/μL primers (Forward primer63F; SEQ ID NO: 1, Reverse primer15R; SEQ ID NO: 2), 0.5 units of DNA Taq polymerase (Takara Ex Taq hotstart), 1 uL of 10 ng/mL template. The PCR reaction was carried out according to 94°C, 20 seconds, (94°C, 20 seconds, 55°C, 20 seconds, 72°C, 90 seconds)×30cycles, 72°C for 3 minutes. The 16S-rDNA base sequence of the isolate was determined by the dye termination method. The primer used for the alignment reaction was 520R (SEQ ID NO. 3). Homology comparisons were performed with bacterial species sequences registered in the DNA Data Bank of Japan, and those with more than 98% similarity were regarded as the same species. The primers used are listed in Table 4. In the 16S-rDNA of eubacteria and archaea, there are about 10 highly conserved areas at both ends or in the interior. Using primers designed according to the sequences of 63F, 15R, and 520R, genes can be identified regardless of the type of bacteria. Zoom in or analyze sequences.

【Table 4】

Primer properties

For the 241 strains selected by Test Examples 2 and 3, the results of using 16S-rDNA base sequences to determine the biological types of bacteria showed that the biological types of Streptococcus bacteria were mainly determined as Streptococcus oralis, saliva Streptococcus salivarius, Streptococcus mitis, Streptococcus sanguinis, etc. In addition, Lactobacillus gasseri (Lactobacillus gasseri) is the most isolated Lactobacillus bacteria.

Evaluation of test example 5 on tooth surface adhesion

Among the 241 strains selected from the results of Test Examples 2 and 3, all strains were used in the following tests except those strains judged to have safety problems based on the results of the biological type determination test.

The preparation method of the sample and the evaluation of the adhesion to hydroxyapatite (HA) were in accordance with Gibbons et al. hydroxyapatite." Infect. Immun. 1976:14:1109-1112).

Each isolated strain was inoculated into 4 mL of MRS liquid medium, and cultured for 24 hours. After culturing, the cells were centrifuged (1,912×g, 10 minutes, 4.0° C.), and washed twice with PBS. Finally, the bacterial solution with an absorbance of 1.0 when diluted to 550 nm with PBS was used for the adhesion test of HA.

The saliva used in this test was collected in the following manner. After gargling with distilled water, paraffin gum was chewed, and the saliva secreted by the above-mentioned stimuli was collected in a centrifuge tube. In order to inactivate proteolytic enzymes, the mixture was heated at 60°C for 30 minutes, and then centrifuged (10,000×g, 10 minutes, 4.0°C) to obtain a supernatant. Equal amounts of saliva from 5 individuals were mixed, sterilized by filtration (0.22 μm), and stored at 4°C before use.

After 5 mg of hydroxyapatite beads (Bio-Rad 40 μm) were washed with PBS, 4 mL of heated saliva was added and shaken at 37° C. for 30 minutes. After shaking, the beads washed twice with PBS were used as saliva-treated HA (S-HA). In addition, beads prepared under the same conditions after adding PBS instead of saliva were used as PBS-treated HA (P-HA).

Add 2mL of bacterial solution into S-HA and P-HA, shake at 37°C for 1 hour. In addition, as a control, only the bacterial solution was shaken under the same conditions. After the reaction, let stand at room temperature for 10 minutes, and collect 1 mL of the supernatant in a new test tube. In order to dissolve the tiny HA, add 100 μL of 0.1M EDTA to the supernatant, stir and let stand at room temperature for 1 hour. Thereafter, the absorbance at 550 nm was measured, and the binding rate (%) was calculated according to the following formula (1), that is, the proportion of bacteria bound to the HA beads.

【Number 1】

The results are shown in Tables 5 and 6. Among the 241 strains selected by Test Examples 2 and 3, 76% of the strains classified as Streptococcus adhered to S-HA. In addition, Lactobacilli in general have relatively low adhesion to tooth surfaces (see H.J.Busscher. "In vitro Adhesion to Enamel and inViVo Colonization of Tooth Surfaces by Lactobacilli from a Bio-Yoghurt" Caries Res1999:33:403-404. ), the table is arranged in the order of high adhesion of Lactobacillus fermentum, Lactobacillus gasseri, etc. to S-HA.

【table 5】

Comparison of Adhesion Rates of Different Species of Isolated Strains (Streptococcus) to S-HA

a: mean ± standard deviation

【Table 6】

Comparison of Adhesion Rates of Different Species of Isolated Bacteria (Lactobocillus) to S-HA

a: mean ± standard deviation not implemented N.D

Evaluation of Test Example 6 Adhesion of Oral Cells

Among the 241 strains selected from the results of Test Examples 2 and 3, the strains considered to have safety problems were excluded based on the results of the biotype determination test, and the rest were used in the following tests.

1) Culture of cell lines

HO-1-N-1 cells (JCRB0831: cells derived from squamous cell carcinoma of human buccal mucosa, hereinafter abbreviated as "HO") and HSC-3 cells (JCRB0623: cells derived from squamous cell carcinoma of human tongue, hereinafter (abbreviated as "HSC") were cultured under the conditions of 5.0% CO2 ₂ and 37°C. DMEM medium containing 10% bovine fetal serum (GIBCO Company 11885) was used for HO cells, and DMEM/F12 medium containing 10% bovine fetal serum (GIBCO Company 11320) was used for HSC medium.

2) Evaluation of cell adhesion

Use medium containing 10% fetal bovine serum for suspension to make the cells reach 1.0×10 ⁵ cells/mL. This suspension was added to a Lab-Tek II chamber slide (Nalge Nunc) in an amount of 200 μL per compartment, and cultured for 48 to 96 hours. Thereafter, cells not attached to the surface of the glass slide were washed and removed using RPMI1640. After culturing in MRS liquid medium for one night, the number of viable bacteria was adjusted to OD ₆₆₀ =0.25, and 200 μL was added to each compartment, and incubated at 37°C for 10 minutes. Then, the cells were washed 3 times with RPMI1640 to remove bacteria that did not adhere to the cells. After Gram staining of the attachments on the slides, the number of adhered cells per 0.16 ^mm2 contained in them was measured using a light microscope. Measurements are made from arbitrarily selected 6 viewing angles.

The results are shown in Tables 7 and 8. A total of 10 or more bacteria were adhered to HO cells and HSC cells of 7 strains of Lactobacillus and 17 strains of Streptococcus relative to the cells contained in 0.16 mm ² .

Any one of Lactobacillus crispatus YIT12319, Lactobacillus fermentum YIT12320, Lactobacillus gasseri YIT12321 and Streptococcus mitis YIT12322, each unit area has a bond of 30 More than one, with strong adhesiveness.

【Table 7】

Adhesion of isolated strains (Lactobacillus genus) to oral cavity cells

【Table 8】

Adhesion of isolated strains (Streptococcus genus) to oral cavity cells

Evaluation of Test Example 7 on Inhibition of Proliferation of Halitosis-causing Bacteria (Periodontal Pathogenic Bacteria)

Based on the results of the above test examples, the strains shown in Tables 10 and 11 were selected. As the target strain, used the

Porphyromonas gingivalis ATCC33277, Prevotella intermedia ATCC25611, Aggregatibacter actinomycetemcomitans Y4, Streptococcus mutans ( Streptococcus mutans) ATCC25175, cousin Streptococcus (Streptococcus sobrinus) ATCC33478.

The isolated bacterial strain is carried out in the MRS liquid culture medium of 4.0mL to carry out a platinum ear inoculation. After culturing for 24 hours, centrifugation (1,912×g, 10 minutes, 4° C.) was performed to obtain a supernatant. Further, the supernatant was filtered using a 0.22 μm filter, and the filtrate was used as a test sample. The antibacterial activity of each sample was measured by agarose dispersion antibacterial assay (Radial diffusion assay). Add 100 μL of the BHI culture solution of each target strain to 10 mL of TS medium (Tryptic soy broth (Difco) 6.0 mg, Tween 20 2.0 μL, agarose 100 mg, H ₂ O 10 mL) and then solidify in the dish after stirring well. After the medium was solidified, a hole with a diameter of 2.5 mm was opened, and 5.0 µL of a test sample was added thereto. After culturing at 37°C for 1 hour, TS medium (Tryptic soy broth (Difco) 0.6 mg, agarose 100 mg, H ₂ O 10 mL) was overlaid and cultured at 37°C. The culture conditions are listed in Table 9. After the cultivation, the diameter of the clear area (inhibition zone diameter) formed outside the wells in which bacteria did not proliferate was measured according to the following formula (2).

Antibacterial ring diameter (mm) = net area diameter of test sample (mm) - hole diameter (mm) (2)

【Table 9】

Culture conditions and positive controls for measuring proliferation-inhibiting effects

The results are shown in Tables 10 and 11. Experiments have shown that in the culture supernatant of Lactobacillus in the isolated strain, it has a strong effect of inhibiting the proliferation of Porphyromonas gingivalis. Moreover, it also has the effect of hindering the proliferation of Prevotella intermedia and Synobacteria actinomycetes. Since in this test there were strains that had an inhibitory effect on Porphyromonas gingivalis even when the pH of the culture supernatant was adjusted to 7.0, it was considered that these strains were likely to produce Antibacterial substances such as hydrogen peroxide or bacteriocins. In addition, it was found that Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 has the effect of inhibiting the proliferation of Streptococcus mutans (Streptococcus mutans and cousin Streptococcus sobrinus) belonging to dental caries pathogenic bacteria. As a result of the test after adjusting the pH of the culture supernatant to 7.0, no effect of inhibiting proliferation was found. Therefore, we believe that Streptococcus mutans and cousin Streptococcus sobrinus produce acid, and bacteria It has acid resistance itself, so it is less likely to inhibit proliferation by lowering the pH value. Therefore, we believe that Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 may produce antibacterial substances such as hydrogen peroxide or bacteriocin.

As for the Streptococcus genus, we found that the culture supernatants of 15 strains also had an effect of inhibiting the growth of the bacteria causing bad breath (periodontal pathogenic bacteria), but the strength of the effect was weaker than that of the isolates of the Lactobacillus genus. Since the proliferation of Porphyromonas gingivalis may be hindered even after adjusting their pH value to 7.0, we think that, like Lactobacillus isolates, they have the ability to produce antibacterial substances such as hydrogen peroxide or bacteriocins. possibility.

In Lactobacillus crispatus YIT12319, Lactobacillus fermentum YIT12320, Lactobacillus gasseri YIT12321 and Streptococcus mitis YIT12322, the total value of the inhibition ring diameter (diameter) All reached more than 9 mm, showing a strong proliferation-inhibiting effect.

【Table 10】

Proliferation-inhibiting effects of isolated strains (Lactobacillus genus) on halitosis-causing bacteria (periodontal pathogens) and dental caries bacteria

*Ss; S.sobrinus ATCC33478, Sm; S.mutans ATCC25175, Pg; Pgingivalis ATCC33277, Pi; Pintermedia ATCC25611, Aa; Adjust the pH to 7.0

【Table 11】

Proliferation-inhibiting effects of isolated strains (Streptococcus genus) on halitosis-causing bacteria (periodontal pathogens) and dental caries bacteria

*Ss; S.sobrinus ATCC33478, Sm; S.mutans ATCC25175, Pg; Pgingivalis ATCC33277, Pi; Pintermedia ATCC25611, Aa; Aggregatibacter actinomycetemcomitans Y4, (-); The pH of the supernatant was adjusted to 7.0

Test Example 8: Evaluation of Pathogenicity of Infective Endocarditis Using Rat Experimental Endocarditis Samples

Based on the results of the above test examples, the strains shown in Tables 12 and 13 were selected. Lactobacillus rhamnosus (Lactobacillus rhamnosus) YIT0227, a pathogenic bacterium of endocarditis, was used as a positive control.

As test animals, 102 Crl:CD (SD) male mice (Charles River, Japan, Inc.) at the age of 8 weeks were used.

Experimental endocarditis (non-bacterial endocarditis) animal samples were prepared in the following manner. As an anesthetic, ketamine injection (containing 50 mg/mL of ketamine hydrochloride) and xylazine (containing 20 mg/mL of xylazine hydrochloride) were mixed at a ratio of 18:5, and then injected into the abdominal cavity at a dosage of 2 mL/kg. Next, under anesthesia, the skin of the neck was incised to expose the right carotid artery, and a 0.61 mm outer diameter polyethylene tube (Natsume, SP10) was inserted through the right carotid artery and placed in the left ventricle. On the second day after making the animal with non-bacterial thrombotic endocarditis, use a 1mL disposable syringe and a 27G injection needle to inject the bacterial solution into the tail vein at an equivalent of 0.5mL/mouse.

The bacterial solution to be injected was prepared as follows. First, 10 μL of isolates were inoculated in 4 mL of MRS medium, and cultured at 37° C. for 20 hours. Then 100 μL of the culture solution was inoculated into 10 mL of fresh MRS medium, and cultured at 37° C. for 20 hours. After the cultivation, the culture solution was centrifuged (1,670×g, 10 minutes, 4.0° C.) to remove the supernatant. Add the same amount of physiological saline as the culture medium, thoroughly stir and wash by vortex, and centrifuge again (1,670×g, 10 minutes, 4.0°C) to remove the supernatant. After the above cleaning operation was repeated three times, physiological saline was added to dilute to the standard bacterial count (4.0-9.0×10 ⁶ CFU/mL), and used as the bacterial solution for input.

Peripheral venous blood collected 4 days after the test strain was put into use was diluted in sections with physiological saline, and 0.5mL of the stock solution and 1.0mL of the 10 ^-2 dilution were applied to the MRS plate medium by the mixed release method. After culturing at 37°C for 3 days, count the colonies propagated on the plate, and calculate the number of viable bacteria in 1 mL of blood. Similarly, the heart homogenate containing warts was diluted in sections with physiological saline, and 1.0 mL of the original solution, 10 ^-2 , 10 ^-4 , and 10 ^-6 dilutions were spread on the MRS plate medium by the mixed dilution method. After culturing at 37°C for 3 days, the colonies propagated on the plate were counted, and the number of viable bacteria was calculated. If no colony is detected in the two test dishes, it is assumed that one colony is obtained from any one of the test dishes, and the value obtained by dividing the weight of the wart by 1 is used as the limit value of detection.

The results are shown in Tables 12 and 13. In the mice injected with positive control YIT0227, 5 out of 6 cases (1-28 CFU/mL) from blood, and all specimens from warts (1.4×10 ³ ～1.7×10 ⁶ CFU/heart) Bacteria were detected in all of them. On the other hand, in mice administered with Lactobacillus crispatus YIT12319, Lactobacillus crispatus LBS17-11, Lactobacillus fermentum YIT12320, and Lactobacillus gasseri YIT12321, from No input bacteria were detected in blood or warts. Therefore, it was determined that these strains were negative for endocarditis pathogenicity in mice.

In addition, in mice administered with Streptococcus mitis YIT12322, the administered bacteria were not detected in the blood, and only one sample bacteria was detected in the warts, so it was judged as negative. In mice administered with other strains, the injected bacteria were detected from all individual warts, and the number of detected bacteria was relatively high (6.2×10 ⁴ to 9.4×10 ⁶ CFU/heart), so all the strains were rejected. judged to be positive.

【Table 12】

Pathogenicity test of an isolated strain (Lactobacillus genus) using mouse samples for infective endocarditis

A: The average number of bacteria in the tested individual, b: Below the detection limit

【Table 13】

Pathogenicity test of an isolated strain (Streptococcus genus) using mouse samples for infective endocarditis

a; The average number of bacteria in the tested individual

Evaluation of Test Example 9 on Dental Caries Using Artificial Oral Devices

Lactobacillus crispatus YIT12319, Lactobacillus fermentum YIT12320, Lactobacillus gasseri YIT12321, Streptococcus mitis YIT12322, Lactobacillus crispatus LBS17- 11 and Lactobacillus gasseri (Lactobacillus gasseri) LBS46-52. In addition, Streptococcus sobrinus 6715 and Streptococcus sobrinus ATCC33478 were used as positive controls.

After the fresh TS medium was inoculated with 3 platinum fungus in the test tube with the isolated strain stored or in the test tube with 4mL MRS medium, the anaerobic culture was carried out at 37°C for 16 hours. Inoculate 4mL of this culture solution into 1L of fresh TS or MRS medium, and perform anaerobic culture at 37°C for 16 hours. The culture solution was centrifuged (4,830×g, 20 minutes, 4° C.) to collect bacteria. After washing it with PBS, the bacterial cells separated by centrifugation again were suspended in 200 mL of PBS. A part of it was sampled and diluted to 1/10 with PBS, and its turbidity was measured at 540 nm, and the whole was adjusted to OD540=0.5 or 1.0 with PBS.

The changes in the hardness of bovine tooth enamel caused by the bacterial solution were tested using an artificial oral device. Specifically, the change of pH value with time was tested by a pH meter installed under the artificial biofilm. The hardness change of bovine tooth enamel is obtained through the Vickers (Vickers) hardness change of the teeth before and after the experiment. Then immerse each bovine tooth enamel slice in 0.5N NaOH (2mL) at 0°C for 10 minutes to separate the artificial biofilm and perform centrifugation (4,830×g, 20 minutes) to separate the bacteria and supernatant liquid (water-insoluble dextran division). According to the value obtained by measuring the OD540nm turbidity after the bacteria are suspended in PBS, the amount of bacteria per ^mm2 tooth piece is obtained. In addition, the amount of water-insoluble dextran in the supernatant was measured by the phenol sulfuric acid method.

For Lactobacillus gasseri (Lactobacillus gasseri) YIT12321, the cariogenicity of the artificial oral device was evaluated under the condition that the bacterial cell concentration and the reaction time were doubled, respectively.

The results are shown in Figures 1-4. Streptococcus sobrinus 6715, a cousin of dental caries pathogenic bacteria, was exposed to the bacteria in the presence of sucrose for 20 hours, and the hardness of the enamel of bovine teeth decreased by about 60 to 80% compared to before the start of the test (Fig. 1, 3 ). In addition, since water-insoluble glucan and bacterial cells were recovered from the enamel (Fig. 2), it is considered that the bacteria formed a biofilm mainly composed of water-insoluble glucan on the surface of the enamel, and the acid produced in this Promotes the loss of enamel, thereby reducing the hardness.

On the other hand, the hardness reduction ratio of Lactobacillus crispatus YIT12319, Lactobacillus fermentum YIT12320, Lactobacillus gasseri YIT12321 and Streptococcus mitis YIT12322, any strain Both are less than 5% (Figure 1, 3). And no water-soluble glucan was recovered from the enamel produced by any strain (Fig. 2, 4).

As for Lactobacillus gasseri (Lactobacillus gasseri) YIT12321, the results of a cariogenicity test conducted using an artificial oral device under the conditions of doubling the bacterial cell concentration and the reaction time are shown in Fig. 5 . Since the reduction rate of enamel hardness is basically the same (4.4%) compared with the original condition, it can be considered that the possibility of inducing dental caries is relatively low even if the bacteria is excessively ingested.

The sucrose assimilation property of Lactobacillus gasseri (Lactobacillus gasseri) YIT12321 was investigated in order to investigate the reason why the hardness of bovine tooth enamel does not decrease. The sucrose assimilation used was ILS liquid medium (BBL Trypticase Peptone (BD) 10g, Yeast Extract (BD) 5g, Bacto Tryptose (BD) 3g, KH ₂ PO ₄ 3g, K ₂ HPO ₄ 3g, (NH ₄ ) ₃ C ₆ H ₅ O ₇ 2g, Lactose 20g, L-cysteine HCl 0.2g, CH ₃ COONa 1g, Tweeen 801g, Salt solution (MgSO ₄ 7H ₂ O 11.5g/100mL, FeSO ₄ 7H ₂ O 0.68g/100mL, MnSO 4.5H ₂ O (2.4 g/100 mL) 5 mL, DW 1000 mL) lactose was replaced with sucrose _, and cultured at 37° C. for 24 hours. The growth of bacteria was expressed by the absorbance of the culture solution measured by OD660nm.

The results showed that Lactobacillus gasseri (Lactobacillus gasseri) YIT12321 did not have a substrate for the growth of sucrose production ( FIG. 6 ). Since the standard strain of Lactobacillus gasseri (YIT0192T) can assimilate sucrose, this property can be considered to be strain-specific.

From the above results, it can be judged that the isolated strains of this test are Lactobacillus crispatus YIT12319, Lactobacillus fermentum YIT12320, Lactobacillus gasseri YIT12321 and Streptococcus mitis YIT12322 , will not cause dental caries. In particular, YIT12321 is considered to be the most useful strain because it does not assimilate sucrose.

YIT12319, YIT12320, YIT12321 and YIT12322 belong to new strains, they are Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500), Lactobacillus fermentum (Lactobacillus fermentum) YIT12320 (FERM BP-11501), Lactobacillus gasseri (Lactobacillus The names of gasseri) YIT12321 (FERM BP-11502) and Streptococcus mitis YIT12322 (FERM BP-11503) have been deposited in the Patent Biodepositary Center of the Independent Administrative Agency Product Evaluation Technology Foundation (Address: East Tsukuba City, Ibaraki Prefecture) 1-1-1 Central No. 6).

The results of Test Examples 6 and 9 regarding the strains that obtained good results in the above Test Examples 2, 3 and 5 (the strains that have no ability to produce VSC and water-insoluble glucan, and have adhesion to the tooth surface) (List) as shown in Table 14. The present inventor has concentrated research on about 1600 strains of microorganisms isolated from the oral cavity, and found only 4 strains of lactic acid bacteria with all properties of (1) to (6) of the present invention, and confirmed that the lactic acid bacteria with this specific quality are in the It is extremely useful in aspects such as food and beverages and oral compositions for preventing or improving various oral diseases or discomforts.

【Table 14】

① Adhesiveness: The result of Test Example 6 (Total cells/0.16mm ² ) was 30 or more...○, less than 30...×

②Inhibition of proliferation: The result of Test Example 7 (total diameter of the antibacterial ring) is 9mm or more...○, 9mm or less...×

③Pathogenicity: The result of Test Example 8 (pathogenicity of infective endocarditis) was negative...○, positive...×

④Hardness change: Results of Test Example 9 (hardness change of bovine tooth enamel) 5% or less...○, 5% or more...×

Verification of the effect of test example 10 on improving the environment in the human oral cavity

In order to investigate the effect of ingesting the lactic acid bacterium Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 of the present invention on the oral environment, 17 subjects with bad breath were taken as objects, and they were allowed to ingest 3 capsules once a day for 4 consecutive weeks. Test food containing more than 3.3×10 ⁸ cfu live bacteria per grain.

Stimulated saliva (saliva secreted after chewing paraffin gum for 5 minutes) was collected from the test subjects' mouths on the start day (week 0) of the test food intake period and the fourth week of intake, and the following oral bacterial counts were measured.

The amount of periodontal pathogenic bacteria (PCR invasion method), the amount of dental caries pathogenic bacteria (culture method), the amount of lactic acid bacteria (culture method)

In addition, in terms of clinical parameters, the following items were measured on the intake start day and the fourth week of intake. Gingivitis index and bleeding at the time of detection were measured by a dentist.

evaluation item

1) Gingivitis index (GI: Lee & Sillness, 1963 reform)

Evaluation benchmark

0: Clinically normal gingiva

1: Although there was mild inflammation and a slight color change of the gums, no bleeding was found caused by rubbing the probe against the inner edge of the gums.

2: There is moderate inflammation, the gums are red with swelling and luster, and bleeding caused by rubbing against the inner edge of the gum margin is found.

3: Severe inflammation, with obvious redness, edema, natural bleeding, and ulcer formation.

Diagnostic site

A total of 24 tooth surfaces on the tongue side, lip side, cheek side, proximal side, and distal side of the following 6 teeth.

【Table 15】

Evaluation method

The average gingivitis score per test subject was calculated from the total score of the diagnostic site using the following formula.

【Number 2】

2) Bleeding during detection (BOP)

Evaluation benchmark

0: No bleeding found

1: found bleeding

Diagnostic site

There are 24 tooth surfaces in total on the tongue side, lip side, cheek side, proximal side, and distal side of the following 6 teeth.

【Table 16】

Evaluation method

Using the following formula, the average bleeding score at the time of detection was calculated for each subject from the total score of the diagnostic site.

【Number 3】

For all statistical analyzes of human trials, statistical analysis software (SAS Preclinical Application Package Ver. 5.0, SAS Institute Inc.) was used. The significance level of each statistical treatment is 5%. The value at the 4th or 8th week of ingestion was tested for statistical significance with respect to the value at 0th week, and the variation during the test period was analyzed.

Table 17 shows the test results of the number of bacteria in the saliva of the 17 subjects. The bacterial counts of Porphyromonas gingivalis, Fusobacterium nucleatum, and Tannerella forsythia, which are periodontal pathogenic bacteria that cause bad breath, were taken in the fourth week after ingesting the lactic acid bacteria There was a decrease (P=0.019, 0.006, 0.005), and the number of lactic acid bacteria increased (P=0.001) at the 4th week of ingesting the lactic acid bacteria. Streptococcus mutans did not change during the test period.

【Table 17】

*: P<0.05, **: P<0.01

That is, by ingesting the lactic acid bacteria of the present invention, periodontal pathogenic bacteria (Porphyromonas gingivalis, Fusobacterium nucleatum, Tannerella forsythia, etc.) ), the bacterial count of ) decreased, and it was confirmed that this lactic acid bacterium is extremely useful as a preventive and/or therapeutic agent for oral diseases and bad breath. Furthermore, the number of caries-causing Streptococcus mutans did not change during the test period. Therefore, it can be considered that the possibility of an increase in the risk of dental caries due to ingestion of the lactic acid bacteria of the present invention is low.

In addition, the bleeding and gingivitis indexes at the time of detection showed significantly lower values in the 4th week after ingestion of the lactic acid bacteria than at the start of ingestion. The analysis results of the evaluation items are shown in Table 18.

【Table 18】

*: P<0.05. **P<0.01

That is, it has been confirmed that the ingestion of the lactic acid bacteria of the present invention improves the clinical parameters of periodontal diseases such as gingivitis, and it has been confirmed that the lactic acid bacteria are extremely useful as a preventive and/or therapeutic agent for oral diseases and bad breath.

Test Example 11 Validation of the Halitosis Suppressive Effect of Porphyromonas gingivalis on Positive Subjects

In order to investigate the effect of ingesting the lactic acid bacterium Lactobacillus crispatus YIT12319 of the present invention on bad breath, 7 people were tested positive for Porphyromonas gingivalis that produced bad breath inducing substances (one of periodontal pathogenic bacteria). For 8 consecutive weeks, let them ingest the test food containing 3.3×10 ⁸ cfu or more live bacteria per 1 capsule according to the amount of 3 capsules once a day for 8 consecutive weeks, and then use gas chromatography to analyze the concentration of volatile sulfur compounds (VSC). Measurements were taken.

Table 19 shows the VSC concentration analysis results of the Porphyromonas gingivalis positive subject group. As a result of the test of the VSC concentration, in terms of H ₂ S, the 4th week and the 8th week of ingestion of the lactic acid bacteria showed significantly lower values than those at the start of ingestion. In addition, the Total VSC showed a significantly lower value in the 8th week of ingestion of the lactic acid bacteria than at the start of ingestion.

【Table 19】

*: P<0.05, **: P<0.01

That is, it has been confirmed that the lactic acid bacteria of the present invention are actually effective in reducing halitosis-causing substances, and it has been confirmed that the lactic acid bacteria are extremely useful as a preventive and/or therapeutic agent for oral diseases and bad breath.

Design and synthesis of test example 12 primers

Using the RAPD method to compare the genomic DNA of 17 strains of Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 and other Lactobacillus crispatus (Lactobacillus crispatus) and found the specificity of the target strain (Lactobacillus crispatus (Lactobacillus crispatus) YIT12319) in three parts DNA sequence. Then these base sequences were interpreted, the primer candidate groups shown in Table 20 were selected, and 35 primer pairs specific to Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 were designed by appropriately combining them.

【Table 20】

Primer name sequence Lc(X)2a gcacatcgttatagtgaacggcgc (serial number 4) Lc(X)2b cacatcgttatagtgaacggcgct (serial number 5) Lc(X)2c acggttcaatacttctaacacatccgc (serial number 6) Lc(X)2d acacatccgcttgatct tgt tgttc (serial number 7) Lc(X)3a ttggttgggttaccgtcaac (serial number 9) Lc(X)5a ctttcactagtggagtgtatttac serial number 10) Lc(X)5b gtctttcactagtggagtgtatttac (serial number 11) Lc(X)5c ctttcactagtggagtgtatttactc (serial number 12) Lc(X)3b gttgacggtaacccaaccaa (serial number 13) Lc(X)5d gtaaatacactccactagtgaaag (serial number 14) Lc(X)5e gtaaatacactccactagtgaaagac (serial number 15) Lc(X)5f gagtaaatacactccactagtgaaag (serial number 16) Lc(X)6a ttaaggtctatggaaacaactccaa (serial number 17) Lc(X)7a gtagaaaccaagttttaaggtctatg (serial number 8)

Test Example 13 selects specific primer pairs with respect to the same species (Lactobacillus crispatus)

Aiming at the genomic DNA of 20 strains of Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 and other Lactobacillus crispatus (Lactobacillus crispatus), 35 kinds of primer pairs designed were used for PCR reaction test. As a result, among the four primer pairs shown in Table 21, DNA amplification products by PCR were confirmed only in Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (Table 21, FIG. 7 ).

Here, Lc(X)2a is represented by sequence number 4, Lc(X)2b is represented by sequence number 5, Lc(X)2c is represented by sequence number 6, Lc(X)2d is represented by sequence number 7, Lc(X) 7a is represented by sequence number 8.

【Table 21】

Test example 14 selects the specific primer pair for 27 genus 143 bacterial strains except lactobacillus crispatus (Lactobacillus crispatus)

Using the four primer pairs shown in Table 21, 65 bacterial strains of the Lactobacillus genus other than Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 and Lactobacillus crispatus (Lactobacillus crispatus) shown in Table 22, 7 bacterial strains closely related to the genus Lactobacillus, oral cavity The genomic DNA of 22 genera and 71 strains (a total of 27 genera and 143 strains) of the priority bacteria was tested by PCR reaction. As a result, among all the primer pairs, the DNA amplification product was confirmed only in Lactobacillus crispatus (Lactobacillus crispatus) YIT12319.

Combined with the results of Test Example 13, 4 primer pairs that did not respond to 163 strains of 27 genera but only responded to Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 were obtained.

【Table 22】

Species investigated for specificity

Manufacture of Test Example 15 Tablet

The ingredients (per 1 tablet) shown in Table 23 were mixed, and the mixture was made into tablets.

【Table 23】

The tablets thus obtained are stable and easy to take.

【Table 24】

The tablets thus obtained are stable and easy to take.

Claims (27)

1. A preventive and/or therapeutic agent for oral diseases, comprising Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri and Streptococcus mitis One or two or more lactic acid bacteria selected from among the following (1) to (6) as active ingredients, (1) Does not have the ability to produce volatile sulfur compounds (VSC) (2) Does not have the ability to produce water-insoluble dextran (3) Adhesive to tooth surface and/or oral cavity cells (4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria (5) No pathogenicity of infective endocarditis (6) No caries. 2. The preventive and/or therapeutic agent for oral diseases as claimed in claim 1, wherein the lactic acid bacteria are obtained from Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500), Lactobacillus fermentum (Lactobacillus fermentum) YIT12320 (FERM BP-11501), Lactobacillus gasseri (Lactobacillus gasseri) YIT12321 (FERM BP-11502) and Streptococcus mitis (Streptococcus mitis) YIT12322 (FERM BP-11503) selected one or more species. 3. The preventive and/or therapeutic agent for oral diseases according to claim 1 or 2, wherein the oral diseases are periodontal diseases. 4. An agent for preventing and/or improving bad breath, which is selected from Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri and Streptococcus mitis One or more than two kinds of lactic acid bacteria that have all the following properties (1) to (6) are used as active ingredients, (1) Does not have the ability to produce volatile sulfur compounds (VSC) (2) Does not have the ability to produce water-insoluble dextran (3) Adhesive to tooth surface and/or oral cavity cells (4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria (5) No pathogenicity of infective endocarditis (6) No caries. 5. The halitosis prevention or improvement agent as claimed in claim 4, wherein the lactic acid bacteria are obtained from Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500), Lactobacillus fermentum (Lactobacillus fermentum) YIT12320 (FERM BP-11501) 1 or more selected from Lactobacillus gasseri YIT12321 (FERM BP-11502) and Streptococcus mitis YIT12322 (FERM BP-11503). 6. A lactic acid bacterium named as Lactobacillus crispatus (Lactobacillus crispatus) YIT12319, submitted for preservation as FERM BP-11500; or named as Lactobacillus fermentum (Lactobacillus fermentum) YIT12320, as FERM BP-11501 Lactic acid bacteria submitted for preservation; or lactic acid bacteria named as Lactobacillus gasseri (Lactobacillus gasseri) YIT12321, submitted for preservation as FERM BP-11502; or named as Streptococcus mitis YIT12322, as FERM BP -11503 Lactic acid bacteria submitted for deposit. 7. A food or drink comprising the lactic acid bacteria according to claim 6. 8. The food and drink according to claim 7, which is a fermented product. 9. An oral composition comprising the lactic acid bacteria according to claim 6. 10. The use of a preventive and/or therapeutic agent for oral diseases, which is to use Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus fermentum (Lactobacillus fermentum), Gardner One or more lactic acid bacteria selected from Lactobacillus gasseri and Streptococcus mitis and having all of the following properties (1) to (6), (1) Does not have the ability to produce volatile sulfur compounds (VSC) (2) Does not have the ability to produce water-insoluble dextran (3) Adhesive to tooth surface and/or oral cavity cells (4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria (5) No pathogenicity of infective endocarditis (6) No caries. 11. The use as claimed in claim 10, wherein the lactic acid bacteria are obtained from Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500), Lactobacillus fermentum (Lactobacillus fermentum) YIT12320 (FERM BP-11501), milk One or more species selected from Lactobacillus gasseri YIT12321 (FERM BP-11502) and Streptococcus mitis YIT12322 (FERM BP-11503). 12. The use according to claim 10 or 11, wherein the oral disease is periodontal disease. 13. The use of a halitosis prevention and/or improvement agent in the manufacture of the halitosis prevention and/or improvement agent, using Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri (Lactobacillus gasseri) and Streptococcus mitis selected from one or more kinds of lactic acid bacteria having all the following properties (1) to (6), (1) Does not have the ability to produce volatile sulfur compounds (VSC) (2) Does not have the ability to produce water-insoluble dextran (3) Adhesive to tooth surface and/or oral cavity cells (4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria (5) No pathogenicity of infective endocarditis (6) No caries. 14. The use as claimed in claim 13, wherein the lactic acid bacteria are obtained from Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500), Lactobacillus fermentum (Lactobacillus fermentum) YIT12320 (FERM BP-11501), milk One or more species selected from Lactobacillus gasseri YIT12321 (FERM BP-11502) and Streptococcus mitis YIT12322 (FERM BP-11503). 15. A lactic acid bacterium for preventing and/or treating oral diseases, which is obtained from Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri and Streptococcus mitis lactic acid bacteria selected from mitis) and having all the following properties (1) to (6), (1) Does not have the ability to produce volatile sulfur compounds (VSC) (2) Does not have the ability to produce water-insoluble dextran (3) Adhesive to tooth surface and/or oral cavity cells (4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria (5) No pathogenicity of infective endocarditis (6) No caries. 16. The lactic acid bacteria as claimed in claim 13, wherein the lactic acid bacteria are obtained from Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500), Lactobacillus fermentum (Lactobacillus fermentum) YIT12320 (FERM BP-11501), milk One or more species selected from Lactobacillus gasseri YIT12321 (FERM BP-11502) and Streptococcus mitis YIT12322 (FERM BP-11503). 17. The lactic acid bacteria according to claim 15 or 16, wherein the oral disease is periodontal disease. 18. A lactic acid bacterium used for preventing and/or improving bad breath, which is obtained from Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus gasseri and Streptococcus mitis ) and have one or more than two kinds of lactic acid bacteria selected from (1) to (6) below, (1) Does not have the ability to produce volatile sulfur compounds (VSC) (2) Does not have the ability to produce water-insoluble dextran (3) Adhesive to tooth surface and/or oral cavity cells (4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria (5) No pathogenicity of infective endocarditis (6) No caries. 19. The lactic acid bacteria as claimed in claim 18, wherein the lactic acid bacteria are obtained from Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500), Lactobacillus fermentum (Lactobacillus fermentum) YIT12320 (FERM BP-11501), milk One or more species selected from Lactobacillus gasseri YIT12321 (FERM BP-11502) and Streptococcus mitis YIT12322 (FERM BP-11503). 20. A method for preventing and/or treating oral diseases, characterized in that, Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus gasseri (Lactobacillus gasseri) and Streptococcus mitis ( Streptococcus mitis), and have the following (1) ~ (6) all the properties of one or more effective amount of lactic acid bacteria allocated to the patient, (1) Does not have the ability to produce volatile sulfur compounds (VSC) (2) Does not have the ability to produce water-insoluble dextran (3) Adhesive to tooth surface and/or oral cavity cells (4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria (5) No pathogenicity of infective endocarditis (6) No caries. 21. The method as claimed in claim 20, wherein the lactic acid bacteria are obtained from Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500), Lactobacillus fermentum (Lactobacillus fermentum) YIT12320 (FERM BP-11501), milk One or more species selected from Lactobacillus gasseri YIT12321 (FERM BP-11502) and Streptococcus mitis YIT12322 (FERM BP-11503). 22. The lactic acid bacterium according to claim 20 or 21, wherein the oral disease is periodontal disease. 23. A method for preventing and/or improving halitosis, characterized in that, Lactobacillus crispatus (Lactobacillus crispatus), Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus gasseri (Lactobacillus gasseri) and Streptococcus mitis mitis) and have the following (1) to (6) all the properties of one or more than two kinds of lactic acid bacteria to patients, (1) Does not have the ability to produce volatile sulfur compounds (VSC) (2) Does not have the ability to produce water-insoluble dextran (3) Adhesive to tooth surface and/or oral cavity cells (4) It has the effect of hindering the proliferation of bad breath-causing bacteria and/or periodontal pathogenic bacteria (5) No pathogenicity of infective endocarditis (6) No caries. 24. The method as claimed in claim 23, wherein the lactic acid bacteria are obtained from Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500), Lactobacillus fermentum (Lactobacillus fermentum) YIT12320 (FERM BP-11501), milk One or more species selected from Lactobacillus gasseri YIT12321 (FERM BP-11502) and Streptococcus mitis YIT12322 (FERM BP-11503). 25. A primer or probe directed to a nucleotide sequence selected from SEQ ID NO: 4 to 8 or Lactobacillus crispatus YIT12319 (FERM BP-11500) formed by arranging complementary nucleotide sequences on the nucleotide sequence ) is specific. 26. A pair of primers directed against a base sequence selected from SEQ ID NO: 4 and 8, SEQ ID NO: 5 and 8, SEQ ID NO: 6 and 8, or SEQ ID NO: 7 and 8 or by complementing each other on the base sequence Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500) formed by the arrangement is specific. 27. A detection method for Lactobacillus crispatus (Lactobacillus crispatus) YIT12319 (FERM BP-11500) characterized by using the primers, primer pairs or probes described in claim 25 or 26.